Metabolic engineering employs recombinant DNA technology to restructure metabolic networks of microorganisms leading to improved production and yields of natural products (Bailey, 1991) This method alters a synchronous series of transformations, defined as a pathway, to produce metabolites. Such pathway manipulations require an awareness of inherent complex regulation and a comprehensive understanding of the discrete enzymatic transformations involved. Metabolic engineering recently emerged in response to efforts made towards improving cellular function by modifying and/or introducing specific biochemical processes (Stephanopoulos, 1996).
Examples of the utility of metabolic engineering have been described and include a modified Candida utilis strain, a diploid yeast, and a modified E. coli strain, each altered to produce carotenoids (e.g., tetraterpenes). Miura et al. (1998b) and Yamano et al. (1994) describe the engineering of Escherichia coli to produce the tetraterpene lycopene by introducing recombinant Erwinia uredovora crtE, crtB and crtI genes.
Miura et al. (1998a) described a Candida utilis strain that produces lycopene, β-carotene, and astaxantin via an engineered carotenoid biosynthetic pathway that included recombinant Erwinia uredovora crtE, crtB, crtI, crtY, crtZ and crtW genes modified to contain the preferred codon usage for Candida and then expressed under the control of constitutive promoters. This strain demonstrated 0.4 mg–1.1 mg lycopene per gram dry weight of cells. Employing a similarly modified C. utilis strain comprising recombinant carotenoid biosynthetic genes from E. uredovora, Shimada et al. (1998) co-expressed the C. utilis HMG-CoA reductase catalytic domain to yield 4.3 mg lycopene/g dry weight of cells. Adding a heterozygous squalene synthase deletion, ERG9, in the same strain effected lycopene production at 7.8 mg lycopene/g dry weight of cells was produced. Wang et al. (1999) engineered Escherichia coli to generate geranylgeranyl pyrophosphate by overexpressing concomitantly E. coli isopentenyl diphosphate isomerase and Archaeoglobus fulgidus GGPP synthase. The cells were further modified to contain the Agrobacterium aurantiacum crtBIYZW gene cluster to produce the carotenoid astaxanthin. U.S. Pat. No. 5,589,581, and EP Patent Nos. EP0769551 and EP0393690, are directed to Erwinia uredovora DNA sequences which encode enzymes that participate in carotenoid biosynthesis.
U.S. Pat. No. 5,429,939 and EP Patent No. 0769551 are directed to a process for producing geranylgeranyl pyrophosphate by transforming a host with a DNA sequence consisting of an Erwinia uredovora enzyme involved in carotenoid biosynthesis and which effects transformation of farnesyl pyrophosphate (FPP) to geranylgeranyl pyrophosphate (GGPP).
Terpenes are compounds derived from isopentenyl pyrophosphate and represent a vast and structurally diverse group of natural products comprising at least 30,000 compounds displaying more than 300 ring systems. Terpenes perform crucial roles in vertebrates and include the retinoids, the geranylgeranyl and farnesyl moiety of prenylated proteins, the coenzymes A, vitamins A, D and E, cholesterol and the steroid hormones. Similarly, terpenoid hormones and pheromones are important in invertebrates. Plants control growth and development using regulatory terpenes including the gibberellins, the brassinosteroids, and abscissic acid. Many plants synthesize defense terpenoids that interfere with biological processes in potential herbivores. Some of these compounds are medicinally useful, such as Taxol, ginkgolide and artemisinin.
One terpene sub-class is the diterpenes. In plants, diterpenes serve as defense toxins, volatile defensive signals, pollinator attractants, and photoprotectants (Bohlmann et al., 1998; McGarvey and Croteau, 1995). In addition to the physiological utility imparted to their host, some diterpenes have exhibited clinical and medicinal relevance, such as the diterpene glycosides found in Pseudopterogorgia elisabethae that demonstrate anti-inflammatory activity (Look et al., 1986; Mayer et al., 1998). Generally, commercial diterpene production often begins with extraction from natural sources followed by, if necessary, synthetic manipulation. However, natural sources are limited and commercial-scale total syntheses are usually impractical. Therefore, an alternative source for the efficient and inexpensive production of diterpenes is lacking in the art.
The present invention is directed to providing a terpene, specifically a diterpene, producing system in a unicellular organism. In one embodiment a haploid S. cerevisiae strain produces significant yields of diterpene and diterpene precursors and is particularly useful as a production mechanism for these compounds.